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Elucidating the Active δ-Opioid Receptor Crystal Structure With Peptide and Small-Molecule Agonists


Elucidating the Active δ-Opioid Receptor Crystal Structure With Peptide and Small-Molecule Agonists

Tobias Claff et al. Sci Adv.


Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current "opioid crisis." Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects.


Fig. 1
Fig. 1. Thermostabilized DOP construct without the N-terminal fusion protein, with both agonists KGCHM07 and DPI-287, used for crystallization, and effects of the crystal structure construct point mutations on the pKi values of the agonists.
The binding affinities (pKi) of KGCHM07 (orange) and DPI-287 (blue) on membrane preparations of HEK cells expressing WT or mutant DOP constructs were determined by their ability to inhibit the binding of [125I]-deltorphin I, used as a selective radioligand. Data were analyzed using a nonlinear fitting analysis, and the Ki values were calculated using GraphPad Prism 7.0. Ki values in the competition studies were determined from IC50 values using the Cheng-Prusoff equation and are represented as means ± SEM of three to six independent experiments, each performed in duplicate. Differences (delta) in pKi values compared to WT are shown. The statistical significance was determined using a nonparametric one-way analysis of variance (ANOVA), showing that all pKi differences of crystal construct mutants versus WT were statistically not significant (P > 0.05).
Fig. 2
Fig. 2. Activation-related changes in the DOP.
Comparison of conserved activation microswitches of the active-like DOP-KGCHM07 (orange) and DOP-DPI-287 (blue) structures with the inactive DOP-naltrindole structure (yellow, PDB 4N6H). Structural superposition of the (A) overall architecture, (B) PIF motif, (C) NPxxY and DRY motifs, and (D) CWxP motif.
Fig. 3
Fig. 3. Effects of sodium-binding mutations on receptor function.
Comparison of the collapsed sodium-binding pocket in DOP-KGCHM07 (orange) with (A) inactive DOP (yellow, PDB 4N6H) and (B) active MOP (purple, PDB 5C1M) with perspective from the extracellular space. Water molecules are shown as blue spheres and the Na+ ion as a yellow sphere. Gi-mediated cAMP signaling of (C) sodium-binding pocket mutants and (D) crystal structure construct mutants with sodium-binding pocket mutations restored to WT residues in response to different KGCHM07 concentrations (signals normalized to WT DOP). β-Arrestin2 recruitment of (E) sodium-binding pocket mutants and (F) crystal structure construct mutants with sodium-binding pocket mutations restored to WT residues in response to different KGCHM07 concentrations (signals normalized to the G95D mutant). Results are expressed as means ± SEM from n = 4 (EPAC) or n = 3 (β-arrestin2) independent experiments, each performed in triplicate.
Fig. 4
Fig. 4. Polar network around D1283.32 and basic amine positioning as potential hallmark for opioid receptor activation.
BRIL-DOP-KGCHM07, orange; BRIL-DOP-DPI-287, blue; naltrindole DOP antagonist structure (PDB 4N6H), yellow; DIPP-NH2 DOP antagonist structure (PDB 4RWD), cyan; DAMGO MOP agonist structure (PDB 6DDF), red. (A) Overview of the KGCHM07 peptide binding pocket. The omit Fo-Fc electron density of KGCHM07 is shown in blue mesh (contoured at 3.0 σ). (B) Overview of the DPI-287 binding pocket. The omit Fo-Fc electron density of DPI-287 is shown in orange mesh (contoured at 3.0 σ). (C) Polar network anchoring the basic amine of DOP agonists. (D) Gi-mediated cAMP signaling of D1283.32 mutants in response to different DOP agonist concentrations (upper panel, KGCHM07; lower panel, DPI-287). (E) Docking poses of DOP agonist peptides (gray) show that all primary amines embedded deeper into the binding pocket (yellow marks), when compared to antagonist DIPP-NH2 (cyan) as indicated by the purple arrow. Similarly, the MOP-DAMGO complex (dark red) is displaced. The cyan arrow indicates related side movements of D3.32. For clarity, only residue one (Phe1 or Dmt1) is depicted, and the surfaces of DOP agonist KGCHM07 and DOP antagonist DIPP-NH2 are shown in orange and green mesh, respectively, to clarify its location in the binding pocket. (F) Docking poses of DOP small-molecule agonists (gray) show all substituted basic amines (N4) that penetrated deeper into the binding pocket, when compared to the antagonist naltrindole (yellow).
Fig. 5
Fig. 5. Activation-related changes in the ECL3 region of the DOP and structural basis for DPI-287 selectivity.
Comparison of ECL3 conformations between (A) inactive (naltrindole, yellow, PDB 4N6H and DIPP-NH2, cyan, PDB 4RWD) and (B) active DOP binding pockets (DOP-KGCHM07, orange; DOP-DPI-287, blue). (C) Alignment of agonist-bound opioid receptor binding pockets. Pocket-forming residues are shown as sticks, with labels indicating Ballesteros-Weinstein nomenclature (22) and red numbers pointing to nonconserved residues. Note that the E6.58 side chain of the KOP is not resolved in the KOP structure. (D) Opioid receptor sequence alignment of the nonconserved ECL3 (light red box) and the region close to the extracellular ends of helices VI and VII. The amino acids of MOP (E312) and KOP (H304) corresponding to DOP’s R291 in the ECL3 region are highlighted in light red.
Fig. 6
Fig. 6. Docking pose of DPI-287–related DOP agonists.
(A) Alignment of the docking pose of the selected DPI-287 analogs BW373U86, SNC-80, and SNC-162 (all gray) with DPI-287 (blue). The blue box indicates the moiety with differences between these three docked analogs. (B) Docking pose of a DPI-287 analog with N-3,4-(methylenedioxy)benzyl substitution (green) and lacking the phenolic hydroxy function into a DOP model derived from the DOP-DPI-287 structure with G952.50D, S1313.35N, and D1082.63K reversed to WT, superimposed with DPI-287 (blue). The surface of the derivative is shown in green, and the black arrow indicates that the ligand is able to penetrate deeper into the entrance of the former sodium-binding pocket. (C) Superposition of the docking poses of DPI-130 (brown) and DPI-3290 (yellow) with DPI-287 suggests that the rotated W2846.58 is essential for DOP binding. (D) Chemical structures and DOP binding properties (human opioid receptors) of (+)-BW373U86, SNC-80, and SNC-162 (29). (E) Chemical structures and binding properties (rat opioid receptors) of DPI-130 and DPI-3290 (32).

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